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A Microfluidic Platform for Trapping, Releasing and Super-Resolution Imaging of Single Cells Ying Zhoua, Srinjan Basub, Kai J

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A Microfluidic Platform for Trapping, Releasing and Super-Resolution Imaging of Single Cells Ying Zhoua, Srinjan Basub, Kai J View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Apollo Accepted Manuscript Title: A microfluidic platform for trapping, releasing and super-resolution imaging of single cells Author: Ying Zhou Srinjan Basu Kai J. Wohlfahrt Steven F. Lee David Klenerman Ernest D. Laue Ashwin A. Seshia PII: S0925-4005(16)30425-7 DOI: http://dx.doi.org/doi:10.1016/j.snb.2016.03.131 Reference: SNB 19936 To appear in: Sensors and Actuators B Received date: 15-12-2015 Revised date: 15-3-2016 Accepted date: 24-3-2016 Please cite this article as: Ying Zhou, Srinjan Basu, Kai J.Wohlfahrt, Steven F.Lee, David Klenerman, Ernest D.Laue, Ashwin A.Seshia, A microfluidic platform for trapping, releasing and super-resolution imaging of single cells, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.03.131 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A microfluidic platform for trapping, releasing and super-resolution imaging of single cells Ying Zhoua, Srinjan Basub, Kai J. Wohlfahrtb, Steven F. Leec, David Klenermanc, Ernest D. Laueb, Ashwin A. Seshiaa,* aNanoscience Centre, Department of Engineering, University of Cambridge, 11 JJ Thomson Avenue, Cambridge CB3 0FF, United Kingdom bDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom cDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom *To whom correspondence may be addressed. Email: [email protected]. Key words: Hydrodynamic trapping; Particle manipulation; Single cell analysis; Super-resolution imaging; Embryonic stem cells. Highlights: Efficient, reliable and long-term trapping of single particles and cells. Selective releasing or retrieving trapped particles/cells. A stable platform for super-resolution imaging at a near molecular resolution. Study of mouse embryonic stem cells using photoactivated localisation microscopy. Identify centromeres of ~ 200 nm size with a precision of < 15 nm. Abstract A multi-layer device, combining hydrodynamic trapping with microfluidic valving techniques, has been developed for on-chip trapping, releasing and manipulation of single cells and particles. Such a device contains a flow layer with trapping channels to capture single particles or cells and a control layer with valve channels to selectively control the trap and release processes. Particles and cells have been successfully trapped and released using the proposed device. The device enables the trapping of single particles with a trapping efficiency of greater than 95%, and has the advantage that single particles and cells can be trapped, released and manipulated by simply controlling corresponding valves. Moreover, the trap and release processes are found to be compatible with biological samples like cells. Our device allows immobilising of large numbers of single cells in a few minutes, significantly easing the experiment setup for single-cell characterisation and offering a stable platform for both single-molecule and super-resolution imaging. Proof-of-concept super- 1 resolution imaging experiments with mouse embryonic stem cells (mESCs) have been conducted by exploiting super-resolution photoactivated localisation microscopy (PALM). Cells and nuclei were stably trapped and imaged. Centromeres of ~ 200 nm size could be identified with a localisation precision of < 15 nm. 2 1 Introduction Super–resolution fluorescence microscopy approaches such as photoactivated localization microscopy (PALM) not only allow the visualisation of subcellular structures at a spatial resolution approaching that of biomolecules, but also allow the study of protein dynamics within individual cells [1]. They allow the 3D imaging of fluorescent proteins in cells with an accuracy far beyond the diffraction limit of visible light. Such super-resolution techniques for visualising tagged molecules within cells place strict requirements upon sample preparation and require target cells to be immobilised over an extended period of time for the highly precise imaging process [2]. For this purpose, long-term single cell trapping is necessary. Due to the capabilities of precisely controlling biological samples, microfluidic systems provide unique opportunities for single cell study. Trapping of single particles and cells has been demonstrated recently in various microfluidic devices [3-8]. Among various cell capture techniques, hydrodynamic trapping relies on a relatively simple fabrication process (e.g. soft lithography) and ease of implementation (no external instrumentation is required such as lasers, transducers or signal generators). Bell et al. have reported such a hydrodynamic trapping device for immobilisation of Schizosaccharomyces pombe cells to facilitate cell super-resolution microscopy. This device enables cell trapping and imaging, but it does not allow the trapped cells to be released or retrieved from the trapping site in a controllable manner. On the other hand, Tan et al. developed a hydrodynamic approach to reliably immobilise large numbers of beads and an optical-based technique for retrieving beads [9, 10]. The trapping process is based on hydrodynamic trapping and the differential fluidic resistance exhibited between the main and bypass channels in the device. Immobilised beads are released by heating the aluminium pattern near the trap with an infrared laser, thereby generating a bubble near the trap to displace the bead into the main flow. However, this release method requires beads being released in a specific order i.e., the bead upstream has to be released first and that located downstream needs to be released last, in order to avoid beads being re-trapped in empty traps downstream. In addition, there are concerns when using such method to release biological cells as the heat generated in the channel may result in harmful effects. Therefore, there is great interest to explore bio-compatible approaches to trap and release particles or cells in a controllable manner. Multilayer microfluidic valving techniques may serve as a solution to this. Microfabricated valves based on multilayer soft lithography were first described by Unger et al. [11] and have been widely used since then [12-15]. A basic microfluidic valve contains two layers: one layer has the fluid to be controlled (flow layer); the other layer contains control channels (control layer). A valve is created when a control channel and a flow channel cross. When the control channel is pressurised, the valve membrane deflects into the flow channel and thus diminishes the size of the flow channel. The flow channel can be completely sealed when sufficient pressure is applied to overcome the elasticity of the elastomer membrane and the fluid pressure in flow layer (back pressure). When the pressure in the control channel is relieved, the elasticity of elastomer causes the valve membrane to spring back to its original position, opening the valve [16]. In this work, we present a microfluidics-based platform for super-resolution PALM imaging of single protein molecules in individual cells. We have combined hydrodynamic trapping with microfluidic valving techniques to accomplish single particle/cell trapping and release. The device described in this work not only enables single cell trapping with high efficiency, but also allows us to 3 release and retrieve cells from the trapping sites in a controllable manner by controlling on-chip valves. Polystyrene particles with different sizes have been used to test the trapping efficiency of devices with different dimensions. The proposed trapping device is able to reliably and automatically capture and hold a large array of cells with a suitably high density, and thus, can be adapted to build a robust and user-friendly platform for super-resolution microscopy. Proof-of-concept experiments with mouse embryonic stem cells (mESCs) have also been conducted to demonstrate applicability as a research tool for single cell handling and super-resolution imaging. 2 Device design The device contains two layers: the flow layer (containing trapping channels) and the control layer (containing valve channels). The schematic diagram of the two-layer device is shown in Fig. 1A. The flow layer (black) contains an array of trapping sites for single particle/cell trapping. In each trapping unit, there are two oppositely facing traps (for particle trapping in both directions). Between the two traps is a middle chamber. The particle/cell capturing process is based on hydrodynamic trapping and the differential fluidic resistance concept. The valve channel in the control layer (red) is right above and crossing the middle chamber in the flow layer, creating an active valve at the crossed area. The crossed area between the two layers forms a very thin PDMS layer (i.e., valve membrane), which controls the opening or closing the flow channel, depending on the applied pressure in the control channel. The microfluidic valve can be activated by pressurising the valve channel in the control layer and deactivated by relieving the pressure in the control layer. When the middle chambers
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